The cellular and molecular origins of extracellular vesicles released by the helminth pathogen, Fasciola hepatica

Abstract

Parasitic helminths secrete extracellular vesicles (EVs) which have potent immunomodulatory effects. Whilst the cargo of EVs has been characterised for many species, we know little about the mechanisms that govern their biogenesis and release. Using antibodies raised against a panel of Fasciola hepatica EV (FhEV) marker proteins, we have identified multiple sites of EV production in the parasite. Discrete immunofluorescence patterns were observed within the gastrodermal cells and tegumental syncytium for different marker proteins whilst the protonephridial (excretory) system and parenchymal-type 2 cells were identified as additional sites of production (or transit) of FhEVs. Ligation was used to mechanically block the oral sucker, excretory pore, or both, to determine the effect on FhEV release from live adult flukes in vitro. This revealed that FhEVs are predominately derived from the gut, whilst the tegument releases EVs to a lesser extent. The data also suggest that the protonephridial system contributes to the small (120K) EV sub-population. Sphingomyelinase (SMase) activity is a key driver of EV biogenesis in mammalian cells and we have previously identified SMases in FhEVs by mass spectrometry. SMase activity associated with isolated FhEVs was susceptible to the chemical inhibitor GW4869 and treatment of adult flukes with GW4869 led to a significant reduction in 120K EV release in vitro suggesting that a ceramidedependent mechanism could drive 120K EV formation. In contrast, the release of the larger 15K EVs was only moderately impacted, indicating that they form independently of SMase activity.Ultrastructural observation of GW4869-treated F. hepatica tissue showed severe disruption to the parenchyma and vacuolation of the tegument, gastrodermal cells and epithelial lining of the excretory ducts. This work establishes that targeted disruption of EV biogenesis and release in helminths is possible and provides proof-of-concept for future studies investigating EV secretion as a target for parasite control.

Evidence of Immune Modulators in the Secretome of the Equine Tapeworm Anoplocephala perfoliata

“…Key secretory proteins involved in immune modulation, host interaction and parasite survival have been identified as part of the ESP as free proteins or as components of EVs for a number of helminths. Annexins, actins, cathepsin proteases, heat shock proteins (HSPs), helminth defense molecules (HDMs), glutathione transferases (GSTs), and fatty-acid binding proteins (FABP) are among a number of such proteins that have been identified from Fasciola hepatica [ 27 , 30 , 45 , 46 , 47 , 48 ], Calicophoron daubneyi (Rumen Fluke) [ 21 , 49 ], Schistosoma japonicum [ 50 ], and Schistosoma mansoni [ 51 ] representing well characterised platyhelminths. Thus, in depth characterisation of helminth secretomes has the potential to uncover host parasite interaction mechanisms.…”

Developmental regulation and functional prediction of microRNAs in an expanded Fasciola hepatica miRNome

“…Furthermore, inhibiting EV release from helminths would conceivably prevent delivery of miRNAs (and other bioactive molecules) to host immune cells. Recently, (Bennett et al, 2020) showed that a chemical inhibitor of EV biogenesis blocked the secretion of EVs from F. hepatica in vitro as initial proof-of-concept for such an approach. One of the major foci for parasite derived miRNAs has been their potential use as diagnostic biomarkers (Mu et al, 2021) We have identified at least 28 EV-derived secreted miRNAs from adult parasites, which could potentially represent PCR detectable indicators of mature fluke infections.…”

Helminth extracellular vesicles: Interactions with the host immune system

“…Again, platyhelminths show differences as EV release is seen directly from their tegument, a syncytium covering their bodies used for both protection and absorption of nutrients, which in nematodes is replaced by a collagenous extracellular cuticle. In the liver fluke F. hepatica , further evidence suggests that in addition to exosomes shed from the tegument (as indicated by their size and the presence of MVBs), most EVs are released through the gut and then oral sucker, with additional output from the protonephridial system through the parasite’s excretory pore ( Bennett et al, 2020a ).…”

“…This correlates to differences in cellular origins of the EV sub-populations; with the larger 15k EVs released from gastrodermal cells lining the gut and 120k EVs seen beneath the gastrodermis, seemingly within the liver fluke excretory system ( De La Torre-Escudero et al, 2019 ). Physically ligating both the oral sucker and excretory pore of F. hepatica blocked the release of the 120k EVs but not the 15k population ( Bennett et al, 2020a ). A large amount of morphological variability is also seen in EVs from F. hepatica produced throughout the parasites lifecycle, from eggs, juveniles and adults ( Sánchez-López et al, 2020 ).…”

“…Drugs can be used to target their specific means of biogenesis. Bennett et al used the chemical inhibitor of neutral sphingomyelinases GW4869 to successfully repress the release of 120 K EVs from F. hepatica in vitro ( Bennett et al, 2020a ). Sphingomyelinases are needed to convert sphingomyelin to ceramide, which drives ESCRT-independent EV biogenesis via membrane curvature ( Verderio et al, 2018 ).…”